MicroRNAs: The Master Regulators of the Breast Cancer Tumor Microenvironment

Breast cancer is the most commonly diagnosed cancer globally and is among the leading causes of cancer deaths worldwide. Breast cancer mortality rates are increasing due to delays in diagnosis, prognosis, and treatment caused by the coronavirus disease 2019 (COVID-19) pandemic. Identification and validation of blood-based breast cancer biomarkers for early detection is a top priority worldwide. MicroRNAs (miRNAs) show the potential to serve as breast cancer biomarkers. miRNAs are small, endogenously produced RNAs that regulate growth and development. However, oncogenic miRNAs also play a major role in tumor growth and can alter the tumor microenvironment (TME) in favor of cancer metastasis. The TME represents a complex network of diverse cancerous and noncancerous cell types, secretory proteins, growth factors, and miRNAs. Complex interactions within the TME can promote cancer progression and metastasis via multiple mechanisms, including oxidative stress, hypoxia, angiogenesis, lymphangiogenesis, and cancer stem cell regulation. Here, we decipher the mechanisms of miRNA regulating the TME, intending to use that knowledge to identify miRNAs as therapeutic targets in breast cancer and use miRNAs as blood-based biomarkers. © Springer Nature Singapore Pte Ltd. 2022.

Genes Involved in miRNA Biogenesis Are Not Downregulated in SARS-CoV-2 Infection.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

Pyroptosis: A Promising Mechanism Linking SARS-CoV-2 Infection to Adverse Pregnancy Outcomes.

Pregnancy is characterized by a delicate immune balance; therefore, infectious diseases might increase the risk of adverse pregnancy outcomes (APOs). Here, we hypothesize that pyroptosis, a unique cell death pathway mediated by the NLRP3 inflammasome, could link SARS-CoV-2 infection, inflammation, and APOs. Two blood samples were collected from 231 pregnant women at 11-13 weeks of gestation and in the perinatal period. At each time point, SARS-CoV-2 antibodies and neutralizing antibody titers were measured by ELISA and microneutralization (MN) assays, respectively. Plasmatic NLRP3 was determined by ELISA. Fourteen miRNAs selected for their role in inflammation and/or pregnancy were quantified by qPCR and further investigated by miRNA-gene target analysis. NLRP3 levels were positively associated with nine circulating miRNAs, of which miR-195-5p was increased only in MN+ women (p-value = 0.017). Pre-eclampsia was associated with a decrease in miR-106a-5p (p-value = 0.050). miR-106a-5p (p-value = 0.026) and miR-210-3p (p-value = 0.035) were increased in women with gestational diabetes. Women giving birth to small for gestational age babies had lower miR-106a-5p and miR-21-5p (p-values = 0.001 and 0.036, respectively), and higher miR-155-5p levels (p-value = 0.008). We also observed that neutralizing antibodies and NLRP3 concentrations could affect the association between APOs and miRNAs. Our findings suggest for the first time a possible link between COVID-19, NLRP3-mediated pyroptosis, inflammation, and APOs. Circulating miRNAs might be suitable candidates to gain a comprehensive view of this complex interplay.

Evaluation of miRNA-16-2-3P, miRNA-618 levels and their diagnostic and prognostic value in the regulation of immune response during SARS Cov-2 infection.

Following the announcement of the pandemic of COVID-19 in December 2019, several studies focused on how to early predict the severity of the disease in symptomatic and asymptomatic patients. Many cytokines including interleukin-6, interleukin-8, and tumor necrotic factors have been concluded as strong indicators for COVID-19 infection. Additionally, miRNAs have been associated with dysregulation in the immune system. The aim of this study are the following: (1) to estimate the level of miRNA-16-2-3P, miRNA-618, IL-8, IL-1ß as predictors for SARS-CoV-2 complications in PCR negative and positive patients; (2) to assess the biological role and effect of these miRNAs on SARS-CoV-2 pathogenicity. Our study showed that the level of IL-1ß had been significantly associated with patient who need hospitalization, also the alteration of the level of miRNA-16-2-3P, miRNA-618 is positively correlated with the admission of these patients and influence the outcomes of SARS-cov-2 infection. Measurement of miRNA-16-2-3P, miRNA-618, IL-1ß could be a good predictor of COVID-19 patient outcome. However the measurement of IL-8 levels during immune responses in the admitted and in ICU patients could have a prognostic value.

CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction.

Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.

IMC-MDA: Prediction of miRNA-disease association based on induction matrix completion.

To comprehend the etiology and pathogenesis of many illnesses, it is essential to identify disease-associated microRNAs (miRNAs). However, there are a number of challenges with current computational approaches, such as the lack of "negative samples", that is, confirmed irrelevant miRNA-disease pairs, and the poor performance in terms of predicting miRNAs related with "isolated diseases", i.e. illnesses with no known associated miRNAs, which presents the need for novel computational methods. In this study, for the purpose of predicting the connection between disease and miRNA, an inductive matrix completion model was designed, referred to as IMC-MDA. In the model of IMC-MDA, for each miRNA-disease pair, the predicted marks are calculated by combining the known miRNA-disease connection with the integrated disease similarities and miRNA similarities. Based on LOOCV, IMC-MDA had an AUC of 0.8034, which shows better performance than previous methods. Furthermore, experiments have validated the prediction of disease-related miRNAs for three major human diseases: colon cancer, kidney cancer, and lung cancer.

The impact of lncRNA-GAS5/miRNA-200/ACE2 molecular pathway on the severity of COVID-19.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for coronavirus disease (COVID-19), potentially has severe adverse effects, leading to public health crises worldwide. In COVID-19, deficiency of ACE-2 is linked to increased inflammation and cytokine storms via increased angiotensin II levels and decreased ACE-2/Mas receptor axis activity. MiRNAs are small sequences of noncoding RNAs that regulate gene expression by binding to the targeted mRNAs. MiR-200 dysfunction has been linked to the development of ARDS following acute lung injury and has been proposed as a key regulator of ACE2 expression. LncRNA growth arrest-specific transcript 5 (GAS5) has been recently studied for its modulatory effect on the miRNA-200/ACE2 axis. OBJECTIVE: The current study aims to investigate the role of lncRNA GAS5, miRNA-200, and ACE2 as new COVID-19 diagnostic markers capable of predicting the severity of SARS-CoV-2 complications. METHODS: A total of 280 subjects were classified into three groups: COVID-19-negative controls (n=80), and COVID-19 patients (n=200) who required hospitalization were classified into two groups: group (2) moderate cases (n=112) and group (3) severe cases (n = 88). RESULTS: The results showed that the serum GAS5 expression was significantly down-expressed in COVID-19 patients; as a consequence, the expression of miR-200 was reported to be overexpressed and its targeted ACE2 was down-regulated. The ROC curve was drawn to examine the diagnostic abilities of GAS5, miR-200, and ACE2, yielding high diagnostic accuracy with high sensitivity and specificity. CONCLUSION: lncRNA-GAS5, miRNA-200, and ACE2 panels presented great diagnostic potential as they demonstrated the highest diagnostic accuracy for discriminating moderate COVID-19 and severe COVID-19 cases.

Plasma-derived exosomal miRNA as potential biomarker for diagnosis and prognosis of vector-borne diseases: A review.

Early disease diagnosis is critical for better management and treatment outcome of patients. Therefore, diagnostic methods should ideally be accurate, consistent, easy to perform at low cost and preferably non-invasive. In recent years, various biomarkers have been studied for the detection of cardiovascular diseases, cerebrovascular diseases, infectious diseases, diabetes mellitus and malignancies. Exosomal microRNA (miRNA) are small non-coding RNA molecules that influence gene expression after transcription. Previous studies have shown that these types of miRNAs can potentially be used as biomarkers for cancers of the breast and colon, as well as diffuse large B-cell lymphoma. It may also be used to indicate viral and bacterial infections, such as the human immunodeficiency virus (HIV), tuberculosis and hepatitis. However, its use in the diagnosis of vector-borne diseases is rather limited. Therefore, this review aims to introduce several miRNAs derived from exosomal plasma that may potentially serve as a disease biomarker due to the body's immune response, with special focus on the early detection of vector-borne diseases.

Saliva microRNA Profile in Children with and without Severe SARS-CoV-2 Infection.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) may impair immune modulating host microRNAs, causing severe disease. Our objectives were to determine the salivary miRNA profile in children with SARS-CoV-2 infection at presentation and compare the expression in those with and without severe outcomes. Children <18 years with SARS-CoV-2 infection evaluated at two hospitals between March 2021 and February 2022 were prospectively enrolled. Severe outcomes included respiratory failure, shock or death. Saliva microRNAs were quantified with RNA sequencing. Data on 197 infected children (severe = 45) were analyzed. Of the known human miRNAs, 1606 (60%) were measured and compared across saliva samples. There were 43 miRNAs with ≥2-fold difference between severe and non-severe cases (adjusted p-value < 0.05). The majority (31/43) were downregulated in severe cases. The largest between-group differences involved miR-4495, miR-296-5p, miR-548ao-3p and miR-1273c. These microRNAs displayed enrichment for 32 gene ontology pathways including viral processing and transforming growth factor beta and Fc-gamma receptor signaling. In conclusion, salivary miRNA levels are perturbed in children with severe COVID-19, with the majority of miRNAs being down regulated. Further studies are required to validate and determine the utility of salivary miRNAs as biomarkers of severe COVID-19.

Synthetic RNA therapeutics in cancer.

Recent advances in the RNA delivery system have facilitated the development of a separate field of RNA therapeutics, with modalities including mRNA, miRNA, antisense oligonucleotide (ASO), siRNA, and circular (circRNA) that have been incorporated into oncology research. The main advantages of the RNA-based modalities are high flexibility in designing RNA and rapid production for clinical screening. It is challenging to eliminate tumors by tackling a single target in cancer. In the era of precision medicine, RNA-based therapeutic approaches potentially constitute suitable platforms for targeting heterogeneous tumors that possess multiple sub-clonal cancer cell populations. In this review, we discussed how synthetic coding and non-coding RNAs, such as mRNA, miRNA, ASO, and circRNA, can be applied in the development of therapeutics. Significance Statement With development of vaccines against coronavirus, RNA-based therapeutics have received attention. Here, we discuss different types of RNA-based therapeutics potentially effective against tumor that are highly heterogeneous giving rise to resistance and relapses to the conventional therapeutics. Moreover, we summarized recent findings suggesting combination approaches of RNA therapeutics and cancer immunotherapy.

miRNAs, from Evolutionary Junk to Possible Prognostic Markers and Therapeutic Targets in COVID-19.

The COVID-19 pandemic has been a public health issue around the world in the last few years. Currently, there is no specific antiviral treatment to fight the disease. Thus, it is essential to highlight possible prognostic predictors that could identify patients with a high risk of developing complications. Within this framework, miRNA biomolecules play a vital role in the genetic regulation of various genes, principally, those related to the pathophysiology of the disease. Here, we review the interaction of host and viral microRNAs with molecular and cellular elements that could potentiate the main pulmonary, cardiac, renal, circulatory, and neuronal complications in COVID-19 patients. miR-26a, miR-29b, miR-21, miR-372, and miR-2392, among others, have been associated with exacerbation of the inflammatory process, increasing the risk of a cytokine storm. In addition, increased expression of miR-15b, -199a, and -491 are related to the prognosis of the disease, and miR-192 and miR-323a were identified as clinical predictors of mortality in patients admitted to the intensive care unit. Finally, we address miR-29, miR-122, miR-155, and miR-200, among others, as possible therapeutic targets. However, more studies are required to confirm these findings.

Transcriptome Profiling in Autoimmune Diseases

Autoimmune diseases are a group of different inflammatory disorders characterized by systemic or localized inflammation, affecting approximately 0.1–1% of the general population. Several studies suggest that genetic risk loci are shared between different autoimmune diseases and pathogenic mechanisms may also be shared. The strategy of performing differential gene expression profiles in autoimmune disorders has unveiled new transcripts that may be shared among these disorders. Microarray technology and bioinformatics offer the most comprehensive molecular evaluations and it is widely used to understand the changes in gene expression in specific organs or in peripheral blood cells. The major goal of transcriptome studies is the identification of specific biomarkers for different diseases. It is believed that such knowledge will contribute to the development of new drugs, new strategies for early diagnosis, avoiding tissue autoimmune destruction, or even preventing the development of autoimmune disease. In this review, we primarily focused on the transcription profiles of three typical autoimmune disorders, including type 1 diabetes mellitus (destruction of pancreatic islet beta cells), systemic lupus erythematosus (immune complex systemic disorder affecting several organs and tissues), and multiple sclerosis (inflammatory and demyelinating disease of the nervous system). © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2014, 2022.

In silico identification of potential miRNAs -mRNA inflammatory networks implicated in the pathogenesis of COVID-19

COVID-19 has been found to affect the expression profile of several mRNAs and miRNAs, leading to dysregulation of a number of signaling pathways, particularly those related to inflammatory responses. In the current study, a systematic biology procedure was used for the analysis of high-throughput expression data from blood specimens of COVID-19 and healthy individuals. Differentially expressed miRNAs in blood specimens of COVID-19 vs. healthy specimens were then identified to construct and analyze miRNA-mRNA networks and predict key miRNAs and genes in inflammatory pathways. Our results showed that 171 miRNAs were expressed as outliers in box plot and located in the critical areas according to our statistical analysis. Among them, 8 miRNAs, namely miR-1275, miR-4429, miR-4489, miR-6721-5p, miR-5010-5p, miR-7110-5p, miR-6804-5p and miR-6881-3p were found to affect expression of key genes in NF-KB, JAK/STAT and MAPK signaling pathways implicated in COVID-19 pathogenesis. In addition, our results predicted that 25 genes involved in above-mentioned inflammatory pathways were targeted not only by these 8 miRNAs but also by other obtained miRNAs (163 miRNAs). The results of the current in silico study represent candidate targets for further studies in COVID-19.Copyright © 2023 Elsevier B.V.

The Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) Is under Post-Transcriptional Control of microRNAs: Analysis of the Effects of agomiRNAs Mimicking miR-145-5p, miR-101-3p, and miR-335-5p.

(1) Background: MicroRNAs are involved in the expression of the gene encoding the chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator); the objective of this short report is to study the effects of the treatment of bronchial epithelial Calu-3 cells with molecules mimicking the activity of pre-miR-145-5p, pre-miR-335-5p, and pre-miR-101-3p, and to discuss possible translational applications of these molecules in pre-clinical studies focusing on the development of protocols of possible interest in therapy; (2) Methods: CFTR mRNA was quantified by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The production of the CFTR protein was assessed by Western blotting; (3) Results: The treatment of Calu-3 cells with agomiR-145-5p caused the highest inhibition of CFTR mRNA accumulation and CFTR production; (4) Conclusions: The treatment of target cells with the agomiR pre-miR-145-5p should be considered when CFTR gene expression should be inhibited in pathological conditions, such as polycystic kidney disease (PKD), some types of cancer, cholera, and SARS-CoV-2 infection.

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents.

Introduction: Oomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors. Methods: To gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species. Results and discussion: Each of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5' base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes.

CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics

Polymerase chain reaction (PCR)‐based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR‐mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point‐of‐care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR‐based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics‐target preamplification‐free detection, microRNA (miRNA) testing, non‐nucleic‐acid detection, and SARS‐CoV‐2 detection‐are also covered.

Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development. © 2022 by the authors.

Effect of miRNAs, Proinflammatory Cytokines and ACE2 in COVID-19 Pathophysiology

Background: Angiotensin-converting enzyme 2 (ACE2) is the main cellular receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and acts as a pro-inflammatory mediator of Coronavirus disease (COVID-19). The clinical outcome of SARS-CoV-2 infection is influenced by the pro-inflammatory mediators. The specific microRNAs (miRNAs) influence the ACE2 expression and are accountable for the increased circulatory pro-inflammatory mediator levels. Thus, host factors play a crucial role in COVID-19 pathophysiology. The pathogenesis of COVID-19 disease is not well understood. Hence we comprehended the role of miRNAs, pro-inflammatory cytokines, and ACE2 genes in COVID-19 pathophysiology. Method(s): We utilized multiple databases, specifically EMBASE, PubMed (Medline), and Google Scholar, for our search. Discussion(s): SARS-CoV-2 genes could be the target of host miRNAs. The miRNAs regulate the expression of ACE2 in various organs, including the kidney, heart, blood vessels, and lung. ACE2 acts as a pro-inflammatory mediator of SARS-CoV-2 associated disease. Pro-inflammatory cytokines (IL-6, IL-1beta, and TNF) have been associated with severe COVID-19 disease. Hence variation in expression of miRNAs would influence the regulation of COVID-19 pathophysiology. The clinical outcomes of COVID-19 are variable which could be linked with the difference in binding of host miRNA to the target genes. Conclusion(s): Correlation of these genes with severe or critical stages of patients will provide bio-markers for the severity of lung inflammation which would be useful in the rapid identification of patients in need of hospital admission. Analysis of the relationship between the miRNAs and ACE2 will be helpful in designing anti-miR therapy for ACE2-related SARS-CoV-2 infection.Copyright © 2021 Bentham Science Publishers.